Circ-INSR therapy protects against cardiac ischemia/reperfusion injury in mice

Background Cardiac ischemia-reperfusion injury (IRI) occurs when blood flow to the heart is restored after a period of ischemia, causing additional damage to heart tissue. This process leads to myocardial cell death, inflammation, and can worsen heart function. Therefore, to explore new and innovative therapeutic approaches to reduce IRI heart dysfunction are still necessary. Circular RNAs (circRNAs) are a a unique type of noncoding RNA with RNA loop structure. This distinctive characteristic makes circRNAs more stable compared to linear RNAs, indicating the high value for development of RNA drug. Purpose In our previous work, we observed that a CircRNA derived from INSR locus (Circ-INSR) can prevent doxorubicin induced cardiotoxocity (DIC). Apart from the DIC,whether Circ-INSR could be the novel theraputic target for other CVDs are still unknown. Thus, in this project, we aim to further understand the function of Circ-INSR in IRI. Methods We generated AAV9-Circ-INSR as well as recombinant Circ-INSR system for the overexperssion of Circ-INSR. To improve the recombinant RNA delivery efficiency in heart, a cardiac targeting peptide modifed lipid nanoparticle (LNP) system was developed and tested in vivo. Echo-cardiograph was used to evaluate the heart function and TTC staining was performed for the analysis of myocardiac infarct area. Furthermore RNA sequencing was utilized for the evaluation of potential mechanism of Circ-INSR in IRI. Results We observed that Circ-INSR levels were significantly reduced in the infarct region under IRI stress, with no changes observed in the border or remote regions. Notably, Circ-INSR overexpression mediated by AAV9 treatment significantly reduced infarct size and the caspase 3 level in the mouse heart. Additionally, Circ-INSR overexpression rescused IRI-induced cardiac dysfunction. Next, RNA sequencing revealed that over 500 genes were regulated by Circ-INSR in infarct area. And DNA replication and Nod-like receptor signaling pathway were the most enriched pathway regarding the KEGG enrichment in infarct area. Furthermore, to enhance the theraputic value of Circ-INSR in IRI, we further developed in vitro transcribed (IVT) Circ-INSR based therapy. We developed a cardiac-enriched LNP system, CMP-LNP, and found that this LNP was more readily absorbed by cardiomyocytes than other cell types in vitro and in vivo. Next, IVT-Circ-INSR could successfully express Circ-INSR in the IRI mouse heart, and TTC staining result indicated that IVT-Circ-INSR reduce infarct region induced by the IRI stress. Conclusions Our findings indicate that Circ-INSR is a key regulator of IRI. Both AAV9 and IVT-CircRNA-mediated Circ-INSR therapies reduce infarct size and improve heart function, suggesting their potential as promising circRNA-based treatments for IRI-induced cardiac dysfunction. Our data provide cutting-edge insights into novel circRNA therapies, particularly IVT circRNA, for CVDs.